This project will examine the cellular and molecular mechanisms that govern the establishment and plasticity of synapses in a model genetic system, the Drosophila neuromuscular junction. These problems will be studied using manipulations at the cellular and molecular level, applied with single cell precision during embryonic and larval development. We have previously characterized several of the cellular events involved in the establishment and growth of the synapse. We are now poised to apply novel tools, developed in the lab, to test hypotheses about the role of electrical activity during neuromuscular development and functional plasticity. The methods include the use "Electrical Knock Out" or EKO ion channels, that suppress electrical activity in specific motoneurons and/or muscle fibers. There are four specific goals in this proposal. First, we plan to expand our repertoire of tools for controlling electrical activity in vivo to include channel constructs enhance electrical activity, providing a complementary set of tools to the EKO constructs. Second, using these methods, several hypotheses about the roles of electrical activity in regulating neuromuscular connectivity and synaptic refinement during embryogenesis and larval development will be tested. Third, the role of neuromuscular activity in regulating the functional plasticity of the synapse will be examined. The latter analysis will focus on the putative orthograde and retrograde signals which are proposed to regulate a form of synaptic homeostasis at the neuromuscular junction. Finally, by focally control electrical activity in specific cells, we propose to carry out a genetic screen to identify the molecules whose functions are involved in regulating synaptic development and plasticity. The many molecular-genetic tools available in Drosophila, combined with vital imaging and electrophysiological analyses, makes this system particularly well-suited to uncover the molecular mechanisms that govern synaptic development and dynamic functional plasticity.